The spin trapping of pyrimidine nucleotide free radicals in a Fenton system.

The reaction of the hydroxyl radical, generated by a Fenton system, with pyrimidine deoxyribonucleotides was investigated by using the e.s.r. technique of spin trapping. The spin trap t-nitrosobutane was employed to trap secondary radicals formed by the reaction of the hydroxyl radical with these nucleotides. The results presented here show that hydroxyl-radical attack on thymidine, 2-deoxycytidine 5-monophosphate and 2-deoxyuridine 5-monophosphate produced nucleotide-derived free radicals. The results indicate that .OH radical attack occurs predominantly at the carbon-carbon double bond of the pyrimidine base. The e.s.r. studies showed a good correlation with previous results obtained by authors who used x- or gamma-ray irradiation to generate the hydroxyl radical. A thiobarbituric acid assay was also used to monitor the damage produced to the nucleotides by the Fenton system. These results showed qualitative agreement with the spin-trapping studies.     

E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents.

The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.     

Sonolysis, radiolysis, and hydrogen peroxide photolysis of pyrimidine derivatives in aqueous solutions: a spin-trapping study

The sonolysis of aqueous solutions of various dihydropyrimidines and substituted pyrimidines was investigated by ESR and spin trapping with the nonvolatile, water soluble spin trap, 3,5-dibromonitrosobenzene sulfonate (DBNBS) and its deuterated analog to examine the possibility of detecting new radicals specifically generated in the high temperature zones produced by collapsing cavitation bubbles. Similar ESR spectra were obtained from sonolysis of argon-saturated aqueous solutions, from uv photolysis of aqueous solutions containing H2O2, and from gamma radiolysis of nitrous oxide saturated solutions, although sonolysis of aqueous solutions leads to the formation of pyrimidine radicals by H atom as well as OH radical addition to the 5,6 double bond of pyrimidines. No evidence for specific new radicals formed in the high temperature regions induced by cavitation could be found. For the reactions of dihydropyrimidines with hydroxyl radicals additional spin adducts could be detected and identified with the spin trap DBNBS compared to 2-methyl-2-nitrosopropane which was used in previous studies; however, for alkylpyrimidines fewer spin adducts were observed. The use of the deuterated analog of DBNBS is helpful for unambiguous radical structure assignment.     

Reactions of the hydrated electron with pyrimidine nucleosides halogenated at the sugar moiety: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by the reactions of hydrated electrons with pyrimidine nucleosides halogenated at the sugar moiety (2'-chloro-2'-deoxyuridine and 2'-chlorothymidine) were studied by e.s.r. and spin-trapping. 2-Methyl-2-nitrosopropane was used as the spin-trap. The usual spin-trapping technique was extended to frozen and deoxygenated systems to avoid contamination of the trapped radicals with side-products by spin-trapping 2-methyl-2-nitrosopropane itself. When this method was applied to 2'-chloro-2'-deoxyuridine, a free radical at the C-2' position of the sugar moiety was spin-trapped together with a free radical at the C-5 position of the base moiety. This indicates that hydrated electrons both add to the base moiety and eliminate halogen anions from the halogenated sugar moiety. In the case of 2'-chlorothymidine, however, only a free radical attributed to H-addition at the C-6 position of the thymine base was observed. No radicals produced by the reaction of hydrated electrons with the halogenated sugar could be spin-trapped.     

ESR study of electron transfer reactions between gamma-irradiated pyrimidines, adriamycin and oxygen

Solid pyrimidine nucleic acid bases (cytosine, thymine, and uracil) were gamma-irradiated (50 KGy) and dissolved in deaerated solutions of adriamycin in water and dimethylsulfoxide (DMSO). Analogous experiments using unirradiated pyrimidines as controls were also performed. In water only gamma-irradiated cytosine showed a reaction with the adriamycin yielding a single ESR peak (g = 2.0033) consistent with the adriamycin semiquinone radical. Since the unirradiated cytosine gave no reaction, the result suggests an electron transfer from cytosine radicals (generated by gamma-radiolysis) to adriamycin. In DMSO the three gamma-irradiated and unirradiated pyrimidines reacted with adriamycin yielding the adriamycin semiquinone radical observed by ESR. These results suggest that in DMSO an electron is transferred to adriamycin from the pyrimidine radicals and from the parent pyrimidine molecules. However, the process is on the order of 10(5) times more efficient for the pyrimidine radicals. Superoxide radicals (O2-.) were formed following addition of oxygen to the deaerated DMSO solutions containing adriamycin semiquinone radicals. O2-. was spin trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results show a possible reaction sequence in which an electron transferred to adriamycin, by pyrimidine radicals and parent pyrimidine molecules, is subsequently transferred to dissolved oxygen.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline nucleic acid constituents and their halogenated derivatives

Free radicals in gamma-irradiated polycrystalline nucleic acid constituents and their 5-halogenated derivatives have been studied by e.s.r. and spin-trapping. After gamma-irradiation at room temperature, the polycrystalline samples were dissolved in aqueous solutions of t-nitrosobutane (tNB) in the absence or presence of oxygen. For many of the nucleic acid constituents, two types of radicals, -C(5)RH-C(6)H- and -C(5)R-C(6)H2-, formed by H-addition to the double bond [-C(5)R=C(6)H-] of the base, were observed, where R is -CH3 or -H. In addition, radicals formed on the sugar moiety were found for some nucleosides. When oxygen was present in the tNB solution, the relative stability of trapped radicals was changed, and thus the presence of more than one radical species could be established. For halogenated bases, the radical produced by H-abstraction from N(1) was observed, and an additional radical species formed by H-addition to the C(6) position was found for 5-fluorouracil. For halogenated nucleosides, the same spectrum was observed in all compounds except the 5-fluoroderivatives, and was assigned to the radicals produced on the sugar moiety. For 5-fluorodeoxyuridine and 5-fluorouridine, the radical formed by H-addition to the C(6) position of the base was observed. In general, the present results are in good agreement with those of previous single crystal studies, but in the case of halogenated compounds other than the 5-fluoroderivatives, it was not possible to spin-trap the alpha-halo radicals which were the most prominent radicals formed from gamma-irradiation of single crystals at room temperature.     

E.s.r. studies of sulphur-substituted pyrimidines. 2-thio-5-carboxyuracil at 77 K.

Single crystals of 2-thio-5-carboxyuracil were irradiated and studied at 77 K with e.s.r. spectroscopy. Five resonances were observed and related to the sulphur atom in the 2 position of the pyrimidine ring. Three of the resonances have been assigned to three conformations of a radical formed by hydrogen abstraction from N1. The principal values for the nitrogen coupling are 9-7, 0-0 and 0-0 gauss. The g tensor principal values are 2-173, 1-997 and 1-990 for the dominant conformation of this radical. Two other radicals could not be identified unambiguously.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

Free radicals and their biological significance: present and future]

Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.     

New evidence that the Alzheimer beta-amyloid peptide does not spontaneously form free radicals: an ESR study using a series of spin-traps

The direct formation of free radicals from Abeta has been suggested to be a key neurotoxic mechanism in Alzheimer's disease (AD). We have explored the possibility of the spontaneous formation of peptide-derived free radicals during the incubation of Abeta 1-40 by ESR spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt (DBNBS) as spin traps. Employing PBN, we observed spectra during the incubation of beta-amyloid peptide, at 37 degrees C, which included adducts of 2-methyl-2-nitrosopropane (MNP), despite rigorous purification of the PBN before incubation. The formation of some of these adducts was found to be enhanced by ambient laboratory light. Our experiments have led us to propose a hypothesis that PBN undergoes hydrolysis and decomposition in the presence of oxidants, which explains the origin of all of the PBN and MNP adducts observed (even when the PBN is highly purified). Hydrogen peroxide, formed during incubation, could play a major role as an oxidant in these experiments. Of the other three spin traps, only DMPO gave (very weak) spectra, but these could be assigned to its hydroxyl radical adduct, formed as an artifact by the nucleophilic addition of water to DMPO, catalyzed by trace levels of iron ions. Thus, while spectra are observed during our experiments, none of them can be assigned to adducts of radicals derived from the peptide and, therefore, our data do not support the suggestion that radicals are spontaneously formed from beta-amyloid peptide.     

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes.

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

Free radical metabolite of uric acid

Uric acid has previously been shown to act as a water-soluble antioxidant. Although the antioxidant activity of uric acid has been attributed to its ability to scavenge free radicals, the one-electron uric acid oxidation product of such a scavenging reaction has not been detected. It order to determine whether a free radical metabolite of uric acid could be formed via one-electron redox processes, we oxidized uric acid with potassium permanganate, horseradish peroxidase/hydrogen peroxide, and hematin/hydrogen peroxide systems. With the use of the rapid-mixing, continuous-flow electron spin resonance technique, we were able to detect the urate anion free radical in all three radical-generating systems. Based on N15-isotopic-labeling experiments, we show that the unpaired electron of this radical is located primarily on the five-membered ring of the purine structure. We were also able to demonstrate that this radical could be scavenged by ascorbic acid.     

Degradation of Nucleic Acid Constituents by Free Radical Systems

Degradation of three nucleic acid bases, uracil, thymine and cytosine, by free radical systems containing ascorbic acid and/or hydrogen peroxide was found to occur when these two reagents were used in combination. Any significant differences in the mode of degradation were not observed among these pyrimidine bases. Ferrous ion added in this system intensified the degree of degradation.

Phosphate bond cleavage of mononucleotides by ascorbic acid—hydrogen peroxide was also demonstrated to occur in a lesser degree than other phosphate esters.

Special device of spectrometric determination of pyrimidine bases in the reaction system was worked out.     

Turnover of carnitine by rat tissues.

A small release of Pi from a diphenylamine-formic acid digest of DNA was detected after elimination of interpurine phosphodiester bonds was complete. Minor components in the DNA digest were identified as pyrimidine oligonucleotides which had lost one terminal phosphate. Isolated pyrimidine tracts released Pi on redigestion with the formic acid-diphenylamine reagent in amounts that increased with the number of nucleotides in the oligonucleotide taken. The oligonucleotides were also partially degraded by the formic acid-diphenylamine reagent and the degradation (2-3% of phosphodiester bonds between consecutive nucleotides) was almost independent of chain length. The cleavage was random with no preference for a phosphodiester bond flanked by particular nucleosides. This minor lack of specificity in the formic acid-diphenylamine-catalysed degradation of DNA can, however, account for the low recoveries of long pyrimidine tracts previously reported. Any analysis of pyrimidine tracts in a DNA molecule should make some correction for this small degree of degradation if exact assignments of the numbers of pyrimidine tracts are to be made.     

Studies on the generation of hydrogen peroxide during some non-enzymic reactions changing the hyaluronic acid molecule.

The effect of catalase on non-enzymic-induced changes in the conformation of hyaluronic acid in a vitreous humour preparation was measured using viscometry. Ascorbate, heavy metal ions, riboflavin or EDTA all lowered the viscosity of hyaluronic acid solutions. These effects could be prevented by the addition of catalase. This suggested that H2 O2 is produced by these compounds and that the resulting change in conformation of hyaluronic acid may be due to peroxyl and hydroxyl attack by the free radicals thus generated.     

Measurement of products from X-irradiated glycylglycine in oxygen-free aqueous solutions.

The product yields in X-irradiated aqueous solutions of glycylglycine (0.05 M and 1.0 M) were measured under deoxygenated conditions. Comparison was made between the results obtained from X- and 60Co gamma-irradiated glycylglycine solutions reported by Garrison, Sokol, and Bennett-Corniea (Radiat. Res. 53, 376-384, 1973). The mechanisms proposed by Garrison et al. were tested by evaluating the stoichiometric relationships. The two intermediate radicals, deamination and H-abstraction radicals, were produced in the initial interactions of glycylglycine with reactive species (e-aq, OH, H) formed in H2O. Although the difference was fairly large at 0.05 M, the production of deamination radicals agreed well with the consumption of the radicals at 1.0 M. The production and the consumption of H-abstraction radicals were within the estimated experimental error in dilute solutions. Among all the products only the G value of aspartic acid decreased with increasing concentration of glycylglycine. This could be attributed to the fact that more acetylglycine is formed at the expense of aspartic acid at 1.0 M than at 0.05 M glycylglycine solutions. Competitive reactions involved with deamination radicals under conditions of homogeneously distributed reactants are discussed to elucidate the radiation chemistry of glycylglycine.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.